Dietary polyunsaturated fat (n-6 and n-3) results in lower high density lipoprotein (HDL) concentrations in nonhuman primates and the type of polyunsaturated fat has opposing effects on HDL subfraction distribution; relative to saturated fat, n-6 polyunsaturated dietary fat raises and n-3 polyunsaturated fat lowers HDL subfractions of intermediate size and density (HDL2a and HDL3a). We hypothesize that the type of lipid surface on nascent HDL particles modulates the size distribution of plasma HDL subfractions through effects on apoprotein binding and conformation and on lecithin:cholesterol acyltransferase (LCAT) activation. To test the hypothesis synthetic phospholipids (PL); differing in fatty acyl composition, and natural PL, isolated from the plasma of nonhuman primates fed diets containing saturated fat vs. fish oil to alter PL distribution (i.e., phosphatidylcholine, lysophosphatidylcholine, sphingomyelin) and fatty acyl composition will be used: Specific Aim 1) singly or in combination with cholesterol in monolayer studies to determine the binding properties of apoA-I and E at different surface pressures and to test the ability of these apoproteins to displace one another from the various types of lipid surfaces; and Specific Aim 2) to form recombinant complexes with cholesterol, apoA-I and/or apoE that resemble nascent HDL in size and chemical composition. These complexes will be subfractionated and characterized with regard to size, chemical composition, number of apoproteins per particle, LCAT reactivity, apoprotein conformation by circular dichroism and fluorescence spectroscopy, particle stability by differential scanning calorimetry (DSC) and lipid fluidity by proton NMR spectroscopy. Major reaction products isolated by ultracentrifugation and column chromatography after LCAT incubation will be characterized in a similar manner. These studies should increase our understanding of the properties of different lipid surfaces that influence HDL subfraction distribution during the maturation of nascent HDL model systems by LCAT. This study has two advantages over previous studies: (1) comparing results from natural PL, modified in distribution and fatty acid composition by dietary fat with those of various, more simplified synthetic PL; and (2) using noninvasive physical techniques (proton NMR, DSC, fluorescence spectroscopy and circular dichroism) as well as 2 different experimental systems (recombinant particles and monolayer studies) to probe the mechanisms of HDL maturation. In addition, emphasis will be placed on the investigation of effects of n-3 fatty acid enrichment in PL species with regard to apoprotein binding to monolayers and recombinant particle structure.